Recent findings lead to a reassessment of the nature and developmental potentiality of tissue stem cells. In vivo plasticity of tissue stem cells is a new phenomena: hematopoietic stem cells (HSCs) have been reported to generate muscle and liver cells on transplantation, and perhaps also contribute to the brain of mice. Muscle stem cells contribute to the hematopoietic system, while neural stem cells contribute to all germ layers when introduced into mouse blastocysts and to multiple tissues following transplantation into adult mice. The origin of the contributing cells and the potentiality of individual stem cells (clonality) remain to established in many of the reported examples of in vivo plasticity. We hypothesize that key lineage-specific regulatory factors, which are normally involved in cell-type specification, may influence tissue stem cell plasticity. Recent in vitro experiments reveal that gain or loss of such factors may lead to retrograde cell differentiation or transdifferentiation. The experimental plan in this RFA application is designed to test the role of such factors in stem cell plasticity and how specific environmental niches may influence in vivo plasticity. Aim 1 will establish the in vivo developmental potential of a hematopoietic committed stem cell population from in vitro differentiated embryonic stem (ES) cells and mouse yolk sac, and also the influence of loss of hematopoietic or muscle regulatory factors on observed plasticity. The clonality of contributing stem cells will be determined by single cell injection and/or by DNA tagging methods. Aim 2 will test the possible involvement of bone marrow colonization or hematopoietic potential in plasticity by inactivating hematopoietic regulatory genes conditionally in HSCs or muscle stem cells. Aim 3 will assess whether expression of a hematopoietic (SCL/tal-1) or myogenic (MyoD) regulatory gene alters in vivo HSC or muscle stem cell plasticity. Aim 4 will evaluate the effects of hematopoietic or muscle regulatory factors on the developmental potential of mesenchymal or neural stem cells. The various approaches used in this work should provide a mechanistic basis for tissue stem cell plasticity, and offer the opportunity in the future to channel differentiation along specific pathways in a directed manner. These strategies may facilitate cell and gene transfer therapies in the future.